Differential cytokine expression in EBV positive peripheral T cell lymphomas.

AIM: To investigate whether specific cytokines are secreted locally at the tumour site in Epstein-Barr virus (EBV) positive peripheral T cell lymphoma (PTCL). METHODS: An RNase protection assay system was used to study the differential expression of 21 cytokines in parallel in eight cases of EBV positive non-nasal PTCL, and compared with 11 EBV negative non-nasal PTCLs and three EBV positive nasal natural killer (NK) cell lymphomas. RESULTS: Among the eight EBV positive cases, interferon gamma (IFN-gamma), lymphotoxin beta (LT beta), interleukin 10 (IL-10), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta 1 (TGF-beta 1), and IL-1 receptor a (IL-Ra) were frequently detectable. IL-15, IL-6, IL-4, IL-1 beta, TNF-beta, and IL-9 were sporadically detectable. Of the frequently detectable cytokines, IFN-gamma and LT beta were commonly detected in the EBV negative cases. For cases with > 50% EBV encoded small non-polyadenylated RNA (EBER) positive cells, IL-10, TNF-alpha, and TGF-beta 1 were detected in three of three cases, and IL-1Ra in two of three cases. For cases with < 20% EBER positive cells, IL-10 was detected in three of five cases, TNF-alpha in two of four cases, but TGF-beta 1 and IL-1Ra were not detected. Interestingly, IL-6 was detected in two of three cases with > 50% EBER positive cells, but only in one of five cases with < 20% EBER positive cells. For comparison, in NK cell lymphomas, IL-10, TNF-alpha, IL-1Ra, and IL-6 were all detectable, but TGF-beta 1 was not detected at all. Immunohistochemical staining revealed IL-10 in many cells; in contrast, EBV latent membrane protein 1 (LMP1) was only found to be positive in isolated cells. CONCLUSIONS: Certain cytokines, such as IL-10 and TNF-alpha, might be expressed preferentially in EBV positive peripheral T cell lymphomas. It is likely that such a cytokine environment enhances EBV infection and contributes towards tumorigenesis.     

Developmental regulation of the cytokine repertoire in human macrophages: IL-1, IL-6, TNF-alpha, and M-CSF

We were interested in the dependence of constitutive and stimulated cytokine secretion on the stage of macrophage (MAC) differentiation in vitro. Elutriation-purified blood MO were cultured up to 28 days and their secretory repertoire was analyzed under adherence conditions at various culture stages. For each of the cytokines tested, interleukin (IL)-1 beta, IL-6, tumor necrosis factor (TNF)-alpha, and macrophage colony-stimulating factor (M-CSF), a different pattern of regulation was observed. During the initial phase of maturation (up to day 7 in culture) within which the characteristics of normal MO to MAC transformation are achieved, M-CSF was the only cytokine to be secreted constitutively. From the LPS-dependent cytokines, IL-1 beta and IL-6 were downregulated whereas TNF-alpha levels increased severalfold. For the release of IL-1 beta, IL-6, and TNF-alpha a synergistic effect of interferon-gamma (IFN-g) and lipopolysaccharide (LPS) was observed. M-CSF release increased until day 7 in culture with LPS being stimulatory for this particular cytokine only during the first days of differentiation. Upon further cultivation of MAC up to 28 days, LPS-induced IL-1 beta levels remained very low, but IL-6 levels increased again reaching that of blood MO, and TNF-alpha continued to rise reaching levels up to 30-fold higher than in blood MO. M-CSF secretion stayed high with LPS even suppressing constitutive secretion. Long-term cultured MAC started to release IL-6 and TNF-alpha also in the absence of a stimulus and, furthermore, became responsive to IFN-g alone. Our data show that the release of each cytokine investigated is differently regulated during maturation. These results document the functional plasticity of human MAC and emphasize the impact that MO to MAC differentiation may have in vivo.     

A synergistic relationship between TNF-alpha, IL-1 beta, and TGF-beta 1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line.

Intestinal epithelial cells are known to secrete a variety of cytokines and may play a role in the immune response at the intestinal mucosa. However, the regulatory mechanisms that govern the secretion of these cytokines are largely unknown. In this report, we have focused on the cytokine interactions that regulate interleukin (IL)-6 secretion by the non-transformed rat small intestinal epithelial cell line IEC-6. Tumour necrosis factor-alpha (TNF-alpha) was found to enhance both IL-6 mRNA expression and protein secretion by the IEC-6 cells. Furthermore, TNF-alpha acted in synergy with either transforming growth factor-beta 1 (TGF-beta 1) or IL-1 beta to greatly enhance IEC-6 cell IL-6 secretion. Although the IEC-6 cells are known to produce TGF-beta, autocrine-secreted TGF-beta was found to have no effect on the elevated IL-6 secretion induced by both TNF-alpha plus IL-1 beta. However, the addition of activated TGF-beta 1 to IEC-6 cultures stimulated with both TNF-alpha and IL-1 beta resulted in greatly elevated levels of IL-6 secretion. Therefore, activated TGF-beta 1 can augment IL-6 secretion stimulated by TNF-alpha and IL-1 beta, either alone or in combination, suggesting that intestinal epithelial cell IL-6 secretion may be under the control of a cytokine network at the intestinal mucosa.     

Interleukin-8, interleukin-6, and soluble tumour necrosis factor receptor type I release from a human pulmonary epithelial cell line (A549) exposed to respiratory syncytial virus.

The release of interleukin-8 (IL-8), interleukin-6 (IL-6) and the soluble forms of the tumour necrosis factor receptor (sTNF-R) from human pulmonary type II-like epithelial cells (A549) after respiratory syncytial virus (RSV) infection was analysed. RSV infection alone induced a time- and RSV dose-dependent IL-8 and IL-6 release from A549 cells. Furthermore, the soluble form of the TNF-RI was also secreted in a time- and RSV dose-dependent fashion. The soluble TNF-RII was not detected in the cell supernatant of infected epithelial cells. The effect of various cytokines [IL-1 alpha/beta, TNF-alpha/beta, IL-3, IL-6, interferon-gamma (IFN-gamma), transforming growth factor-beta 2 (TGF-beta 2)] and colony-stimulating factors [granulocyte (G)-CSF; granulocyte-macrophage (GM)-CSF] on the IL-8 release from A549 cells was also studied. Our data show that the proinflammatory cytokines IL-1 alpha/beta and TNF-alpha/beta induced an IL-8 release in non-infected A549 cells, and increased the IL-8 release of RSV-infected A549 cells synergistically. In addition, IL-3, G-CSF, IFN-gamma and TGF-beta 2, albeit at high concentrations, induced a low IL-8 release from non-infected A549 cells. The enhanced IL-8 secretion rates were accompanied with elevated cytoplasmic IL-8 mRNA steady state levels, as was shown by Northern blot analysis. Cellular co-culture experiments performed with A549 cells and polymorphonuclear granulocytes or peripheral blood mononuclear cells revealed that increased IL-8 amounts were secreted in the co-culture of non-infected as well as RSV-infected cells. The present study suggests a central role for the airway epithelium during RSV infection with regard to cytokine and cytokine receptor release, resulting in a recruitment and activation of inflammatory and immune effector cells. Our data also suggest that paracrine cytokine networks and cell-cell contact are involved in the regulation of IL-8 secretion within the microenvironment of the bronchial epithelium.     

Differential cytokine expression in EBV positive peripheral T cell lymphomas.

AIM: To investigate whether specific cytokines are secreted locally at the tumour site in Epstein-Barr virus (EBV) positive peripheral T cell lymphoma (PTCL). METHODS: An RNase protection assay system was used to study the differential expression of 21 cytokines in parallel in eight cases of EBV positive non-nasal PTCL, and compared with 11 EBV negative non-nasal PTCLs and three EBV positive nasal natural killer (NK) cell lymphomas. RESULTS: Among the eight EBV positive cases, interferon gamma (IFN-gamma), lymphotoxin beta (LT beta), interleukin 10 (IL-10), tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta 1 (TGF-beta 1), and IL-1 receptor a (IL-Ra) were frequently detectable. IL-15, IL-6, IL-4, IL-1 beta, TNF-beta, and IL-9 were sporadically detectable. Of the frequently detectable cytokines, IFN-gamma and LT beta were commonly detected in the EBV negative cases. For cases with > 50% EBV encoded small non-polyadenylated RNA (EBER) positive cells, IL-10, TNF-alpha, and TGF-beta 1 were detected in three of three cases, and IL-1Ra in two of three cases. For cases with < 20% EBER positive cells, IL-10 was detected in three of five cases, TNF-alpha in two of four cases, but TGF-beta 1 and IL-1Ra were not detected. Interestingly, IL-6 was detected in two of three cases with > 50% EBER positive cells, but only in one of five cases with < 20% EBER positive cells. For comparison, in NK cell lymphomas, IL-10, TNF-alpha, IL-1Ra, and IL-6 were all detectable, but TGF-beta 1 was not detected at all. Immunohistochemical staining revealed IL-10 in many cells; in contrast, EBV latent membrane protein 1 (LMP1) was only found to be positive in isolated cells. CONCLUSIONS: Certain cytokines, such as IL-10 and TNF-alpha, might be expressed preferentially in EBV positive peripheral T cell lymphomas. It is likely that such a cytokine environment enhances EBV infection and contributes towards tumorigenesis.     

Effect of intestinal epithelial cell cytokines on mucosal B-cell IgA secretion: enhancing effect of epithelial-derived IL-6 but not TGF-beta on IgA+ B cells

A variety of cell types may be involved in the regulation of IgA secretion at the intestinal mucosa. Intestinal epithelial cells (IEC) are known to have the capacity to secrete several cytokines which may exert an important regulatory effect on local immunoglobulin secretion by mucosal B cells. In this study, we have determined the effect of secreted cytokines from the rat non-transformed IEC-6 cell line on IgA secretion by IgA+ mesenteric lymph node B cells. Four day IEC-6 cell culture supernatants (SN) were found to enhance lipopolysaccharide (LPS) stimulated IgA secretion by 3-fold and this enhancement was determined to be due to IEC-derived IL-6. Interestingly, IEC-derived TGF-beta as well as recombinant human latent TGF-beta1 were found to have no effect on IgA secretion by the IgA+ B cells suggesting that these cells may be insensitive to the latent form of this cytokine. Finally, the addition of a culture SN from a 5 h culture of isolated normal rat IEC which contained high levels of IL-6 also greatly enhanced IgA secretion by LPS stimulated IgA+ B cells. These results suggest that the IEC may be an important source of IL-6 to enhance local mucosal IgA+ B cell responses.     

Seminal plasma induces mRNA expression of IL-1beta, IL-6 and LIF in endometrial epithelial cells in vitro

The influence of seminal plasma on the mRNA expression of cytokines in human endometrial epithelial and stromal cells and the cytokine production of spermatozoa were investigated in vitro. Seminal plasma and spermatozoa were collected from healthy volunteers and were screened by enzyme-linked immunosorbent assay for cytokines. Epithelial and stromal cells from fertile women were cultured on matrigel or polystyrol and incubated with pooled seminal plasma or with transforming growth factor beta1 (TGF-beta1), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), which were found to be significantly concentrated in seminal plasma. Endometrial cytokine expression was analysed by RNase protection assay and supported by RT-PCR. Supernatants of highly purified spermatozoa did not contain detectable levels of IL-1beta, IL-6 and VEGF. Screening of seminal plasma revealed concentrations >10-fold above the serum level for TGF-beta1, IL-8 and VEGF. Incubation of epithelial cells with 0.1, 1 and 10% seminal plasma resulted in concentration-dependant stimulation of IL-1beta, IL-6 and LIF mRNA expression. Maximum stimulation was found in epithelial cells from tissue samples taken in the mid secretory phase. Epithelial mRNA expression of IL-1beta, IL-6 and LIF increased by stimulation with TGF-beta1 and IL-8, but not with VEGF. In conclusion, seminal plasma stimulates expression of pro-inflammatory cytokines in endometrial epithelial cells in vitro. This effect might at least in part be exerted by TGF-beta1 and IL-8, abundantly present in seminal plasma. The in-vivo physiological relevance of these in-vitro studies remains to be determined.     

Intracellular and extracellular cytokine production by human mixed mononuclear cells in response to group B streptococci

Group B streptococci (GBS) are a major cause of severe infection in newborns, pregnant females, and other immunocompromised hosts. Infection often includes septicemia, shock, pneumonia, and respiratory failure. In previous studies, we have reported that GBS induce marked production of tumor necrosis factor alpha (TNF-alpha) by human mononuclear cells. The present study was designed to measure the production of TNF-alpha as well as additional cytokines, including interleukin 1beta (IL-1beta), IL-6, IL-8, IL-12, and gamma interferon (IFN-gamma) but also to determine from what cells and at what time point during incubation with GBS that these cytokines are produced. Mixed mononuclear cells were incubated with heat-killed GBS, media alone, or 1 microg of Escherichia coli lipopolysaccharide (LPS). Brefeldin A was added to each sample prior to staining, which prevented the export of cytokines by the Golgi apparatus. The cells were then stained with the appropriate conjugated antibodies and analyzed by using a flow cytometer. Results indicate that intracellular cytokines appear, in almost all cases, simultaneous to or before secreted proteins are detected. In contrast to the response to LPS, where TNF-alpha, IL-1beta, IL-6, and IL-8 appear almost simultaneously, the human monocyte response to GBS results in the production of TNF-alpha but delayed appearance of IL-1beta, IL-6, and IL-8. The lymphocyte response to GBS was also strikingly different from that to LPS in that both secreted IFN-gamma and IL-12 was detected, while LPS failed to induce production of these critical cytokines. This suggests an important role for TNF-alpha, IFN-gamma, and IL-12 in GBS pathogenesis and/or immunity.     

Cytokine-induced differentiation of IgA B cells: studies using an IgA expressing B-cell lymphoma.

Cytokines such as interleukin-5 (IL-5) and transforming growth factor beta 1 (TGF beta 1) increase IgA production by heterogeneous populations of lipopolysaccharide (LPS)-activated murine B cells. We have used IgA expressing murine B-lymphoma cells CH12.LX.C4.4F10 (4F10) to define the activity of these and other cytokines on IgA secretion at the single-cell level, membrane IgA expression, IgA polymerization and cell growth. IL-5 as well as LPS significantly increases IgA secretion of 4F10 cells, whereas TGF beta 1, a cytokine known to stimulate isotype switching to IgA among surface IgM-bearing B cells, inhibits IgA secretion. When tested alone, IL-1 beta, IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) do not significantly alter IgA secretion. However, there is a synergistic increase in IgA secretion when 4F10 cells are co-stimulated with IL-5 and IL-4, while IFN-gamma inhibits IL-5-stimulated up-regulation of IgA secretion. In parallel with increased IgA secretion after cytokine stimulation, 4F10 cells display less membrane IgA. Increased J-chain steady-state mRNA levels after IL-5 or LPS stimulation are paralleled by increased mRNA levels for secreted IgA, but are not accompanied by alterations in the ratio of monomeric to polymeric IgA. IL-5 and LPS initially stimulated but later inhibited 4F10 cell proliferation suggesting an inverse relationship between proliferation and differentiation in this cell line. 4F10 cells are a useful model for the characterization of discrete aspects of IgA B-cell differentiation, since the secretory and membrane Ig and proliferative responses of this IgA B-cell line to cytokines and LPS appear to parallel those of freshly isolated murine B cells.     

Interferon-gamma modulates messenger RNA levels of c-sis (PDGF-B chain), PDGF-A chain, and IL-1 beta genes in human vascular endothelial cells.

The authors have investigated the effects of cytokines and lipopolysaccharide (LPS) on mRNA levels of c-sis (platelet-derived growth factor (PDGF)-B chain), PDGF-A chain, and interleukin 1 beta (IL-1 beta) genes in human vascular endothelial cells (EC). IL-1, tumor necrosis factor (TNF), and LPS not only enhanced the accumulation of c-sis mRNA, but also induced IL-1 beta gene expression. Interferon-gamma (IFN-gamma), in contrast, suppressed the accumulation of c-sis mRNA profoundly and PDGF-A chain mRNA to a lesser extent. The cytokine, in addition, suppressed the release of PDGF-like proteins by EC, while maintaining the growth of EC. IFN-gamma, however, augmented the levels of IL-1 beta mRNA in cultured EC in association with LPS or IL-1, suggesting that the suppression of c-sis expression was not mediated through modulation of IL-1 gene expression by IFN-gamma. These results raise the possibility that IFN-gamma may play a novel regulatory role in the pathogenesis of vascular diseases such as atherosclerosis and vasculitis.     

Effects of associated cytokines (IL-1, TNF-alpha, IFN-gamma and TGF-beta) on collagen and glycosaminoglycan production by cultured human synovial cells

The production of collagen and glycosaminoglycans (GAG) was studied in cultured human synovial cells exposed to four cytokines, alone or in dual combination, namely interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and transforming growth factor-beta (TGF-beta). Among these cytokines, only TGF-beta (0.1-10 ng/ml) induced a significant and dose-dependent increase of collagen synthesis in a 24-h incubation. This effect was reversed when the factor was associated with either IL-1 beta (100-500 pg/ml), TNF-alpha (1-100 ng/ml) or IFN-gamma (100 U/ml). Except IFN-gamma which clearly inhibits the collagen production, the other cytokines IL-1 and TNF-alpha were not very effective when tested separately, although they generally induced a small reduction in collagen amount. IL-1 beta and TNF-alpha were found to be more efficient than TGF-beta in stimulating the production of GAG by the synovial cells. IFN-gamma exerted an antagonistic effect on the TGF-beta-induced stimulation of GAG synthesis. TNF-alpha and IL-1 beta were shown to have an additive effect on that production. The results indicate that interactions between cytokines present in the inflamed synovial tissue may modulate their respective actions and thus introduce differentials in their effect on collagen and GAG metabolism which are responsible for the alterations of synovial extracellular matrix in rheumatoid arthritis.     

Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells. Differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-gamma.

The elicitation of leukocytes from the circulation to inflamed tissue depends on the activation of both the leukocyte and endothelial cell. In this study we determined the gene expression and secretion patterns for the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in cytokine- and lipopolysaccharide (LPS)-treated cultured human lung microvascular endothelial cells (HLE). HLE constitutively expressed low levels of MCP-1 and IL-8. Treatment of HLE with a variety of cytokines and LPS up-regulated both IL-8 mRNA expression and release of immunoreactive IL-8 with an order of potency tumor necrosis factor-alpha (TNF-alpha) >> IL-1 alpha > LPS, whereas interferon-gamma (IFN-gamma) had no effect on IL-8 mRNA or antigenic levels. However, IFN-gamma, in combination with high doses of IL-1 alpha, resulted in a synergistic increase in IL-8 generation. MCP-1 gene expression and secretion was induced in a dose-dependent manner after IL-1 alpha, TNF-alpha, IFN-gamma, and LPS activation of HLE. IL-1 alpha was the most potent inducer of MCP-1 generation and LPS was relatively ineffective. IFN-gamma, in combination with low doses of IL-1 alpha, resulted in a synergistic increase in MCP-1 generation by HLE. These results demonstrate that although IL-8 and MCP-1 generation by HLE occurs on cytokine treatment, the relative ability of a given cytokine to elicit IL-8 generation is not directly parallel to effects on MCP-1 generation. These data suggest that the regulation of IL-8 and MCP-1 expression exhibit significant differences in their mechanisms. Such differences in the expression of specific chemokines may explain the specific appearance of various leukocytes at sites of inflammation and injury. These data also directly demonstrate that the lung microvascular endothelium contribute to the cytokine network of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.     

Defective macrophage function in neonates and its impact on unresponsiveness of neonates to polysaccharide antigens

Neonates do not respond to thymus-independent (TI) antigens (Ag), making them vulnerable to infection with encapsulated bacteria. The antibody (Ab) response of adult and neonatal B cells to TI Ag requires certain cytokines, which are provided by T cells or macrophages (MPhi). Lipopolysaccharide (LPS) failed to induce neonatal MPhi to produce interleukin (IL)-1beta and tumor necrosis factor alpha (TNF-alpha) mRNA and to secrete IL-1beta, IL-12, and TNF-alpha. However, LPS induced neonates to secrete some IL-6 and three- to fivefold more IL-10 than adults. Accordingly, adding adult but not neonatal MPhi could restore the response of purified adult B cells to trinitrophenol (TNP)-LPS, a TI Ag. Increased IL-10 is causally related to decreased IL-1beta and IL-6 production, as IL-10(-/-) neonatal MPhi responded to LPS by secreting more IL-1beta and IL-6 than wild-type (WT) neonatal MPhi. When cultures were supplemented with a neutralizing Ab to IL-10, WT neonatal MPhi secreted increased amounts of IL-6 and allowed neonatal MPhi to promote adult B cells to mount an Ab response against TNP-LPS. Thus, neonates do not respond to TI Ag as a result of the inability of neonatal MPhi to secrete cytokines, such as IL-1beta and IL-6, probably as a result of an excess production of IL-10. This dysregulated cytokine secretion by neonatal MPhi may be a result of a reduction in expression of Toll-like receptor-2 (TLR-2) and TLR-4 and CD14.     

Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-6 on the production of immunoreactive IL-1 and TNF-alpha by human monocytes.

The effects of GM-CSF, IL-2, IFN-gamma, TNF-alpha and IL-6 on the production of IL-1 (both secreted and cell associated) and TNF-alpha by peripheral blood monocytes were studied. Monocytes were cultured for 20 h in suspension and in serum-free conditions which minimized background stimulation of monokine production. GM-CSF, IL-2 and TNF-alpha directly induced the production of cell-associated IL-1 but little or no IL-1 or TNF-alpha secretion. Combination of GM-CSF with IFN-gamma, IL-2 or TNF-alpha synergistically enhanced IL-1 secretion and had an additive effect on cell-associated IL-1 production. Combination of IL-2 with IFN-gamma or TNF-alpha also synergistically enhanced IL-1 secretion but the effect on cell-associated IL-1 production was less than additive. GM-CSF synergistically enhanced TNF-alpha secretion induced by IFN-gamma but not by lipopolysaccharide. GM-CSF did not enhance TNF-alpha secretion induced by IL-2 or TNF-alpha. In contrast, IL-2 synergistically enhanced TNF-alpha secretion induced by IFN-gamma. These results are discussed in relation to cytokine involvement in rheumatoid arthritis.     

Cytokine mRNA profiles in mononuclear cells in acute aseptic meningoencephalitis.

Cytokines are important modulators of inflammation and immune responses. Using in situ hybridization with radiolabelled cDNA oligonucleotide probes, we studied the expression of mRNA encoding the cytokines gamma interferon (IFN-gamma), interleukin 4 (IL-4), IL-6, IL-10, transforming growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), lymphotoxin, and perforin in mononuclear cells (MNC) from blood and cerebrospinal fluid (CSF) of patients with acute aseptic meningoencephalitis (AM) and from blood of healthy controls. Patients in the acute phase of AM had elevated numbers of IFN-gamma mRNA-expressing cells in the blood compared with that of controls and higher numbers of IFN-gamma mRNA-expressing cells in their CSF compared with that of convalescent-phase patients, which is in accordance with the antiviral effects of this cytokine. Upregulation of IL-4, IL-6, and IL-10 was found in convalescent-phase patients, which is consistent with the longstanding B-cell response found in AM. TGF-beta and perforin were upregulated in both stages of AM, while the numbers of blood and CSF MNC expressing cytokine mRNA of the TNF family (TNF-alpha and lymphotoxin) did not differ between patients with AM and controls. An even higher elevation in CSF was noticed for MNC expressing most of the cytokines, particularly IL-4 and TGF-beta, reflecting the autonomy of the immune response in the CSF. The definition of cytokine profiles in AM, a self-limiting and benign disease, provides a foundation for future comparisons with other infectious and inflammatory nervous system diseases.